This invention relates to a process for producing L-proline. In particular, it relates to a process for the production of L-proline in high yields by cultivation and work-up of a certain type of algae, specifically, Chlorella sp. 580. More particularly, it relates to a process for recovering L-proline from the algae cells without disrupting or destroying the capability of the cells to resynthesize L-proline so that the proline depleted cells can be reused (recycled) to make additional L-proline. Conditions of cultivation, workup, recovery and recycle are defined.
L-proline is a useful amino acid which is used as a medicine and in other applications. In the past, L-proline has been produced mainly by isolation from hydrolysates of proteins or gelatin or by organic synthesis. However, the yield of product obtained by these methods is very low, and the procedures involved are complicated. As a result, L-proline is one of the most expensive amino acids. Therefore, the development of a process for the mass production of L-proline using inexpensive starting materials would be highly desirable.
The fact that L-proline is contained in algae of the genus Chlorella is known. Further, it is known that L-proline is produced by the Chlorella algae in response to the environment in which it is grown, i.e., the sodium chloride content of the growth medium. The relationship between L-proline cell accumulation in Chlorella emersonii and the sodium chloride content of the environment external to the cell has been described in AUST. J. PLANT Physiology (1979) 6,69-79 in which it was reported that in the emersonii species of Chlorella, L-proline production within the cell generally increases as the sodium chloride concentration is increased in the environment external to the cell. However, it appears that only small amounts of L-proline are produced within Chlorella emersonii and, at sodium chloride concentrations higher than 335 mM, the cells plasmolyze.
The fact that Chlorella sp. 580 excrete proline also is known as reported in Limnol. Oceanographer (1965) 10:192-206. However, the excretion levels reported therein also are small.
Methods for producing L-proline by fermentation also are known. For example, British Pat. No. 1,132,036 discloses that L-proline can be produced from mutant strains of Micrococcus glutamicus by fermentation. Also, U.S. Pat. No. 4,224,409 discloses that L-proline can be obtained by culturing a mutant of the genus Brevibacterium, Corynebacterium or Microbacterium. L-proline production by fermentation, however, is somewhat expensive since the sources of carbon for use in the fermentation medium include carbon sources other than CO.sub.2, such as, for example, pentoses, hexoses, dissaccharides, or the like.
Thus, even though it is known that L-proline can be produced both intracellularly and extracellularly by the genus Chlorella, insofar as applicant is aware, nothing has been reported in the literature with respect to the identification of a specific species of Chlorella and a specific method of cultivating said specific organism such that L-proline can be produced intracellularly within the organism in amounts high enough to be considered sufficient to form the basis for an industrial process.
In applicant's co-pending U.S. application Ser. No. 329,226, filed Dec. 10, 1981, entitled Process for the Preparation of Amino Acids, applicant has identified both a specific species of Chlorella, namely Chlorella sp. 580, and a method of cultivating the species to produce L-proline in amounts high enough to be considered sufficient to form the basis for commercial production. According to applicant's process disclosed therein, Chlorella sp. 580 is cultivated under high-intensity illumination in an aqueous growth medium containing a high concentration of sodium chloride of at least 1 M in the final stage of cultivation, in the presence of an adequate supply of carbon, in a depth not exceeding approximatley 20 cm of the aqueous medium, until algae of high L-proline content are obtained. The algae are then harvested and L-proline is thereafter recovered from the algae. While applicant's aforementined process does provide for the accumulation of high amounts of L-proline with Chlorella sp. 580 algae cells (up to approximatley 35% of the cell weight), the time typically required for cell production (cell growth) is approximatley five to seven times that required for the production of L-proline. However, it can be much longer depending on the prevailing conditions such as the amount of available sunlight when culturing is done outdoors. Thus, it would be highly desirable to reduce this ratio. One way to reduce the time contribution of the initial growth period would be to use the same cells to produce L-proline several times thereby avoiding an initial growth period for each new crop of proline produced. In order to achieve cell recycle, however, and thus reduce the time contribution of the initial growth period, the proline-rich cells must be depleted of their proline in a manner which does not destroy their proline-synthesizing capability so that the cells so depleted can be used again to produce additional amounts L-proline.